We studied the effect of epidermal growth factor (EGF) on signal transduction on three cell lines of different origin. A long terminal repeat (LTR)-driven expression vector containing the normal human EGF receptor was introduced into NIH/3T3, NR6 and 32D cell lines by transfection, leading to overexpression of the receptor. In the cell lines overexpressing the receptor, EGF stimulated the rapid formation on inositol polyphosphates, 1,2-diacylglycerol and arachidonic acid, the mobilization of intracellular Ca++ and the activation of protein kinase C. Formation of Ca++-mobilizing inositol (1,4,5)-trisphosphate was very rapid and transient, reaching its peak l5-30 seconds after stimulation. Conversely, the level of inositol (l,3,4)-trisphosphate increased more slowly, but remained elevated up to five minutes. Measurement of intracellular Ca++ concentration by fura 2, revealed both intracellular mobilization and influx from the outside in response to the growth factor. Taken together, these data indicate that the reconstituted human EGF receptor is able to couple to the phosphoinositide-related intracellular signalling machinery. Since EGF was fully mitogenic in the cell lines tested and induced a similar pattern of second messenger formation irrespective of the origin of the cell line, we suggest that inositol lipid and arachidonic acid metabolism might play a crucial role in the transduction of the EGF mitogenic signal.